Yeast extract an ingredient of TYI-S-33 medium devised for axenic cultivation of Entamoeba histolytica has been replaced with a mixture of defined nutrients consisting of adenine, guanine, cytosine, uracil, AMP, ADP, ATP, and NH4Cl fortified with B vitamins. A method has been devised enabling rapid direct axenization of E. histolytica from cultures in which the parasite is grown with a mixed bacterial flora and thus circumvents the intermediate step of growing the amoebae monoxenically prior to axenization. Butyric acid added to the medium used for axenization is a key factor in success of this technique. An improved medium for axenic cultivation of Giardia lamblia has been achieved by means of supplementing TYI-S-33 medium with crude bovine bile preparations containing free and conjugated bile acids. Growth is superior to that attained with TP-S-l currently the medium of choice for this parasite. Modification of a technique devised in this laboratory has enabled cyropreservation of refractory strains of E. histolytica. Success is attributed to the substitution of sucrose for glucose in the cryopreservant, the use of TYI-S-33 for cultivation of the amoebae before and after freezing and the use of plastic vials in the place of glass.